Clinical Subjects and Specimens
Specimens were collected from a total of 7 cases of CMGT recruited at Northeast Agricultural University Animal Hospital. Tissues were identified and classified. All procedures were performed with consent from the animals’ owners and in accordance with the Experimental Animal Control law of Northeast Agricultural University. All animal care and handling procedures were approved by the Ethics Committee of the Animal Hospital of Northeast Agricultural University.
Cell Lines and Cell Culture
The CHMp cell line was isolated from the primary lesion of a 12-year-old female dog with a malignant breast tumor by the Department of Veterinary Science, University of Tokyo, Japan . CHMp cells were cultured in pink high-glucose DMEM (Meilunbio, China, MA0212), supplemented with fetal bovine serum (FBS) (Tianhang, China, 11011-8611) and penicillin-streptomycin-amphotericin B solution (Beyotime, China, C0224-100ml). Cell suspensions were prepared by digesting logarithmic-phase.
CHMp cells with 0.25% EDTA-trypsin (Beyotime, China, C0201-100ml). The cells were then diluted with complete culture medium containing 20% FBS and inoculated into 96-well plates. An inverted microscope was used to identify wells containing single cells, and then the plates were incubated at 37°C and viewed once every 24h. The surviving cells were expanded and exposed to a drug concentration gradient plus drug maintenance method (final cisplatin concentration of 20 μmol/L) for 10 months to obtain the CHMpCIS cell line.
Western Blot Analysis
Cell proteins were extracted with the BCA Protein Assay Kit (Beyotime Biotechnology, P0012S) and separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to an NC membrane, which was blocked and then incubated sequentially with primary and secondary antibodies. Finally, ECL Luminescent Solution (Beyotime Biotechnology, P0018FS) was added for imaging. Quantification was performed using Image J software.
Cell Counting Kit-8 (CCK8) Assay
Logarithmic-phase CHMp and CHMpCIS cells were adjusted to 3×104 cells/mL and transferred to 96-well plates at 100μL/well for culture at 37℃. After 24 h, the culture medium was replaced with medium containing a gradient of cisplatin concentrations, and the cells were again cultured at 37°C for 24 h. The culture medium was then replaced with CCK8 detection solution (Abcam, UK, ab228554), and the cells were incubated at 37°C for 30 minutes. Finally, OD values were recorded using a microplate reader. The half maximal inhibitory concentration (IC50) of cisplatin was calculated according to relative cell survival.
In separate assays, logarithmic-phase CHMp and CHMpCIS cells were adjusted to 1×104 cells/mL and transferred to 96-well plates at 100 μL/well for culture at 37℃. On days 1, 2, 3, 4, or 5, the culture medium was replaced with CCK8 detection solution, the OD value was detected, and the value-increasing curves were plotted.
Cells adjusted to 1×105 cells/mL were seeded in a 6-well plate and cultured in a 37°C incubator. After 48 h, the cell culture solution was collected, and the cells were harvested by digestion with trypsin and centrifugation. Apoptosis was detected by flow cytometry with Annexin V-FITC/PI (Bioss, China, BA00101).
Cells adjusted to 5×104 cells/mL in serum-free culture medium were transferred to the upper chamber of a Transwell apparatus at 100 μL/well (Corning, USA, 3422). During the migration experiment, Matrigel was not added to the upper chamber, whereas for the invasion experiment, the upper chamber was coated with Matrigel in advance. Complete medium containing 20% fetal bovine serum was added to the lower chamber at 600 μL per chamber and the entire apparatus was placed in a 37°C incubator. After migration for 24 h/invasion for 48 h, the cells on the lower surface of the upper chamber were fixed with paraformaldehyde, stained with crystal violet, washed with distilled water, and dried in a fume hood. Cells on transparent plates were photographed under an inverted microscope (×200 magnification). For counting, 5 fields were randomly selected from each group of cell samples.
Mammosphere Formation Assays
Cells were transferred to DMEM/F-12 (Meilunbio, China, MA0214) containing phenol red and supplemented with B-27 supplement medium (Gibco, USA, 17504044), human EGF recombinant protein (Gibco, USA, PHG0311), and human FGF-basic (FGF-2/bFGF) recombinant protein (Gibco, USA, 13256-029). The cells were adjusted to 5×104 cells/mL and inoculated into an ultra-low-adhesion 6-well plate. The mammospheres were cultivated until most had a diameter of >200μm and were subsequently passaged at 1×104 cells/mL to cultivate second-generation mammospheres (Ⅱ MS). Mammospheres with a diameter >75μm and large mammospheres with a diameter >200μm were then counted.
Clone Formation Assay
Cells were seeded in a 6-well culture plate at 500 cells/well. The medium was replaced with fresh medium on day 5. On day 10, the cells were fixed in 4% paraformaldehyde and stained with crystal violet, and the number of colonies was recorded.
Transfection kits, siR-NC and siR-SOX4 were purchased from Ribobio. Transfection was performed according to the manufacturer's instructions.
Reverse-transcription Quantitative PCR (QRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, USA, 15596026). The total RNA concentration was measured with an ultra-micro ultraviolet spectrophotometer (Thermo, USA, ND-ONE-W), and the RNA was reverse-transcribed using a cDNA first-strand synthesis kit (Tiangen, China, KR118) with TB Green II dye (TaKaRa, Japan, RR820A). Finally, a Light Cycler R 480 System (Roche, Basel, Switzerland) was used for quantitative analysis. Each gene was analyzed at least 3 times in each sample, and the average value was calculated. The data were analyzed by the 2-δδCT method. See Supplementary Table 1 for primer sequences.
Cells were seeded onto sterile coverslips placed in 6-well plates, cultured to 80–90% confluence, washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X-100 (Solarbio, China, T8200 ), blocked with BSA (Solarbio, China, T8020), and incubated sequentially with primary and secondary antibodies and DAPI (Solarbio, China, C0060) in the dark. Anti-fluorescence quenching agent was added, and images were acquired with a fluorescence microscope.
Data are presented as the mean ± SD. Statistical comparisons between treated and control groups were performed using Student’s t-test in GraphPad Prism 5, and multiple groups were compared by two-way ANOVA. Differences that are not significant (P>0.05) are not marked; significant differences (0.01＜P＜0.05) are marked with "*"/"#", and extremely significant differences (P<0.01) are marked with "**"/"##".